Congenital infection with human cytomegalovirus (HCMV) occurs in approximately 40,000 infants a year in the United States annually and is the leading viral cause of birth defects. These problems together with the severe disease that HCMV causes in solid organ and hematopoietic stem cell transplant recipients provides a strong impetus for the development of an effective vaccine. However, unlike viruses that are cleared by the host, the ability of HCMV to persist even in the presence of high levels of host immunity suggests that novel approaches are needed for the development of a vaccine. Because results in the murine CMV (MCMV) model have shown that CD8+ T cells specific for an immunodominant antigen are ineffective at limiting viral replication, we speculated that vaccination using these antigens may at best elicit immunity that is not significantly greater than that generated during the natural infection: protection that is ultimately incomplete since the virus persists and can cause recurrent infection and congenital transmission. We hypothesized that the essential viral proteins that are highly conserved among the CMVs may represent a novel class of T cell targets. The rationale is that these genes must be expressed for viral replication, and the high amino acid conservation needed for maintaining protein activity limits immune escape by mutation. Because these antigens have been found to be mostly subdominant during infection with HCMV or MCMV, we hypothesized that priming of T cells against these antigens may result in qualitatively greater protective immunity than the natural infection. To this end, we have recently shown that DNA immunization using either of two conserved, essential genes of MCMV (the DNA polymerase M54 and the helicase M105) protects mice against a subsequent sublethal challenge with virulent virus. We have also developed a DNA prime - whole, inactivated virus boost strategy that elicits both CD8+ T cell responses as well as strong neutralizing antibodies, and we demonstrated that this prime-boost vaccination conferred complete protection against systemic viral challenge and significant protection against mucosal viral challenge. However, since MCMV does not cause infection in utero, studies of the prevention of congenital CMV infection have utilized the guinea pig CMV (GPCMV) model. While the GPCMV vaccines that have been tested in the guinea pig model have provided statistically significant improvements in pregnancy outcomes, no vaccine has conferred complete protection against mortality or infection. In this proposal, we plan to use the GPCMV model of congenital CMV infection to demonstrate "proof-of-principle" that prime-boost immunization with the conserved, essential genes of GPCMV followed by chemically killed GPCMV is protective against congenital GPCMV infection and disease and to provide the justification for pursuing more comprehensive preclinical studies of immunity and protection. The Specific Aims of this proposal are: 1. Determination of the protective efficacy of prime-boost vaccination with a trivalent DNA (consisting of DNA polymerase, helicase, and glycoprotein B genes) followed by inactivated GPCMV in the guinea pig model of congenital infection and disease, and 2. Characterization of the relative contributions to immunity and protection provided by the DNA polymerase and helicase DNAs, the glycoprotein B DNA, and the inactivated virus. The novel aspects of our proposed vaccine are both the choice of the highly conserved essential viral genes for generating T lymphocyte responses in the priming step as well as the use of a whole, killed virus in the boosting step that together can completely protect against systemic CMV challenge. PUBLIC HEALTH RELEVANCE: Congenital infection with human cytomegalovirus (HCMV) occurs in approximately 40,000 infants a year in the United States annually and is the leading viral cause of birth defects. These problems together with the severe disease that HCMV causes in solid organ and hematopoietic stem cell transplant recipients provides a strong impetus for the development of an effective vaccine. However, unlike viruses that are cleared by the host, the ability of HCMV to persist even in the presence of high levels of host immunity suggests that novel approaches are needed for the development of a vaccine against HCMV and other persistent viruses.